Improved vehicles for endosomal escape

Abstract

Variants of the ZF5.3 peptide having Cys substitutions of one or both His residues of the Cys2His2 Zn(II) coordination site, to form Cys3His (CCHC) or Cys4 (AV5.3), respectively, are used in fusions of the peptides with cargo domains, delivery vehicles and delivery methods.

Description

[0001] Improved Vehicles for Endosomal Escape

[0002]

[0001] Government Support Clause

[0003]

[0002] This invention was made with government support under grant number GM134963 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0004]

[0001] Introduction

[0005]

[0002] Proteins able to successfully circumnavigate into the mammalian cell cytosol or internal organelles have enormous unrealized potential as replacement enzymes, gene editing tools, protein interaction inhibitors, and bispecific ligands. The challenge is that not only must these materials reach the appropriate cellular locale fully intact and in therapeutically relevant concentrations, they also must retain activity upon arrival. The question of residual activity is especially critical when delivery involves exposure to the late endocytic pathway, whose acidic lumenal environment denatures and / or degrades internalized materials. It has been estimated that the RNA within FDA-approved SARS-CoV2 vaccines or siRNA therapeutics reach the cytosol with efficiencies significantly less than 10%.1No FDA-approved protein therapeutic acts within the cytosol.

[0006]

[0003] ZF5.3 is a compact, rationally designed mini-protein that delivers proteins to the cytosol or nucleus with high efficiency.2,3Despite its small size (27 amino acids), ZF5.3 is exceptionally stable, with a thermal melting temperature at or above 90˚C at pH 7.4. Indeed, ZF5.3 can be isolated intact from the cytosol of treated cells,2and guides multiple classes of proteins, including enzymes,3–5transcription factors,6nanobodies,7and monobodies5into the cytosol and / or nuclei of living cells. In the best cases, the efficiency of delivery to the cytosol reaches or exceeds 50% to establish nuclear or cytosolic concentrations of 500 nM or higher.2,8,9Studies have shown that ZF5.3 escapes into the cytosol from late endosomes and / or lysosomes in a mechanistically distinct process that demands a fully assembled HOPS complex, a ubiquitous tethering complex that mediates late endosomal fusion.8Notably, endosomal escape of ZF5.3 and covalent ZF5.3-conjugates proceeds without leakage of other intralumenal components,8with little or no detectable endosomal damage, and is especially efficient when the cargo protein is small, intrinsically disordered, or unfolds at a temperature of 35oC or lower.5

[0007]

[0004] Recent studies suggest that the unfolding of ZF5.3 itself is also critical for efficient endosomal escape.9Although ZF5.3 is exceptionally stable at pH 7.4, between pH 4 and pH 5 ZF5.3 unfolds cooperatively in a transition initiated by protonation of a single Zn(II)-bound His residue. The pKaof this His residue corresponds almost exactly to that of the late endosomal / lysosomal lumen, pH 4.6. Evidence that pH-induced unfolding is essential for

[0008] 1 B25-037-2WO endosomal escape of ZF5.3 derives from the observation that a ZF5.3 analog that lacks bound Zn(II) and remains folded at low pH is taken up into the endocytic pathway but fails to efficiently reach the cytosol.9

[0009]

[0005] Despite the promise of ZF5.3 for cytosolic delivery, the environment within the late endocytic lumen is harsh. The acidic pH, which can be as low as pH 4.5, can degrade RNAs and denature proteins, often irreversibly. Denatured proteins are substrates for lumenal hydrolytic enzymes whose role is to regenerate building blocks for the cell. Although certain therapeutic cargoes successfully delivered by ZF5.3 retain measurable activity after exposure to the endolysosomal lumen, including MeCP26and a Bcl-11A-targeted bio-protac,7other desirable proteins are likely to be less robust. We wondered whether we could avoid the detrimental effects of late endosomal pH by fine-tuning the structure of ZF5.3 to facilitate escape from an earlier point along the endocytic pathway, from a compartment whose pH is higher than 4.6.

[0010]

[0006] Relevant Literature includes: US10227384 disclosing ZF5.3.

[0011]

[0007] Summary of the Invention

[0012]

[0008] The invention provides novel variants of the ZF5.3 peptide having Cys substitutions of one or both His residues of the Cys2His2 Zn(II) coordination site, to form Cys3His (CCHC) or Cys4(AV5.3), respectively, fusions of the peptides with cargo domains, delivery vehicles and related methods. In aspects and embodiments the invention provides:

[0013]

[0009] 1. An engineered endosomal escape polypeptide domain comprising a ZF5.3 sequence (YSCNVCGKAFVLSRHLNRHLRVHRRAT; SEQ ID NO:01) having Cys substitutions of one or both His residues of the Cys2His2Zn(II) coordination site, to form a Cys3His (CCHC) or Cys4(AV5.3) a coordination site, respectively.

[0014]

[0010] 2. An engineered endosomal escape polypeptide domain comprising the sequence

[0011] YSCNVCGKAFVLSRHLNRHLRVCRRAT; (SEQ ID NO:03) (CCHC); or

[0015]

[0012] YSCNVCGKAFVLSRHLNRCLRVCRRAT; (SEQ ID NO:02) (AV5.3).

[0016]

[0013] 3. An endosomal escape polypeptide domain herein, covalently or noncovalently bound to a cargo (e.g. a bioactive molecule, label, nucleic acid and polypeptide), wherein the endosomal escape polypeptide domain preferably facilitates endosomal escape to improve cytosolic delivery of the cargo.

[0017]

[0014] 4. A lipid nanoparticle delivery vehicle for delivering a cargo (e.g. a bioactive molecule, label, protein, nucleic acid, or complexes thereof) to a cytosol, comprising an endosomal escape polypeptide domain herein, covalently or noncovalently bound to the cargo.

[0018]

[0015] 5. A pharmaceutical composition comprising a composition or compound herein and a pharmaceutically acceptable excipient, in effective, unit dosage.

[0019] 2 B25-037-2WO

[0016] 6. A method of delivering a cargo to a cytosol, comprising delivering to a cell a composition herein, under conditions wherein the endosomal escape polypeptide domain facilitates endosomal escape of the cargo to the cytosol, optionally further comprising a subsequent step of detecting the delivery of the cargo to the cytosol.

[0020]

[0017] 7. A method of treating a disease or disorder or condition in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a composition herein, optionally further comprising an antecedent step of detecting the disease, disorder or condition, and / or optionally further comprising a subsequent step of detecting a resultant effect or improvement in the disease, disorder or condition.

[0021]

[0018] The invention encompasses all combinations of the particular embodiments recited herein, as if each combination had been laboriously recited.

[0022]

[0019] Description of Particular Embodiments of the Invention

[0023]

[0020] Unless contraindicated or noted otherwise, in these descriptions and throughout this specification, the terms “a” and “an” mean one or more, the term “or” means and / or. It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein, including citations therein, are hereby incorporated by reference in their entirety for all purposes.

[0024]

[0021] Examples: Earlier endosomal escape improves the catalytic activity of delivered enzyme cargo.

[0025]

[0022] Our re-design of ZF5.3 was inspired by the classic bioinorganic chemistry of zinc finger proteins. Like ZF5.3, many zinc finger proteins contain a canonical Cys2His2Zn(II) coordination site.10,11But many others, both natural and designed, contain Cys in place of one (Cys3His) or both (Cys4) His residues.12This change is significant. The pKaof a Zn(II)-bound Cys side chain generally falls between 5.0 and 6.0, approximately 2.0 pH units higher than the pKaof a Zn(II)-bound His residue.13We therefore hypothesized that variants of ZF5.3 with one or more His-to-Cys substitutions would unfold at pH values higher than 4.6. We hypothesized further that if unfolding is truly critical for endosomal escape, the new variant(s) would escape from endosomal compartments formed earlier along the pathway whose lumen are less acidic. Earlier endosomal escape should translate into improved activity of a pH-sensitive cargo protein.

[0026]

[0023] Here we test these ideas through the design of AV5.3, a variant of ZF5.3 in which Zn(II) is bound not by a Cys2His2motif, but instead by a Cys4motif. Like ZF5.3, AV5.3 unfolds cooperatively at low pH, but in this case the pH midpoint occurs at pH 5.4, not 4.6. Despite this 3 B25-037-2WO difference, AV5.3 and AV5.3-protein complexes are taken up by live cells and traffic into the cytosol with virtually the same efficiency as ZF5.3 and analogous ZF5.3-protein complexes. With AV5.3, however, cytosolic trafficking depends not only on the activity of the HOPS complex, but also on the activity of the CORVET complex, whose substrates are earlier (i.e., less acidic) endosomal vesicles. Finally, we observe that earlier escape is associated with substantially improved activity of a delivered pH-sensitive cargo enzyme. Delivery of the pH-sensitive enzyme dihydrofolate reductase (DHFR) as a conjugate to AV5.3 results in substantially higher cytosolic activity than delivery using ZF5.3. Only the AV5.3-DHFR conjugate effectively rescues a genetic DHFR knockout in live CHO cells.

[0027]

[0024] Chemically tuning the pH required for unfolding: AV5.3 unfolds at a higher pH than ZF5.3.

[0028]

[0025] To test whether variants of ZF5.3 containing one or more His-to-Cys substitutions would unfold at pH values higher than 4.6, we prepared two ZF5.3 variants in which the Cys2His2Zn(II) coordination site was replaced by either Cys3His (CCHC) or Cys4(AV5.3) (Table 1). CCHC and AV5.3 were synthesized using solid-phase methods, purified by HPLC, and their identities verified using LC-MS. We then used circular dichroism (CD) spectroscopy to assess and compare the pH-dependent secondary structures of CCHC and AV5.3 with that of ZF5.3. At pH 7.5 and a concentration of 115 µM, the CD spectra of ZF5.3, CCHC, and AV5.3 were qualitatively similar, with pronounced negative ellipticity at 208 nm, as expected for a molecule containing a ββ⍺ zinc finger fold14,15. As found for ZF5.3,9these features depend on the presence of Zn(II) but are independent of temperature between 5 and 95˚C.

[0029]

[0026] To evaluate the influence of pH on secondary structure, CD spectra were recorded at pH values from 2.5 to 7.5. An overlay of the spectra for CCHC and AV5.3 revealed shifts in the primary ellipticity minimum towards shorter wavelengths as the pH decreased from 7.5 to 2.5. Similar changes were previously reported for ZF5.3 and are consistent with a pH-induced loss of structure that was confirmed using NMR.9A plot depicting the change in ellipticity at 208 nm as a function of pH revealed cooperative transitions for both ZF5.3 and CCHC with a transition over 2 pH units, whereas the transition for AV5.3, while still cooperative, was broader. The transition midpoint occurred at pH 4.6 for CCHC and ZF5.3, whereas the transition midpoint for AV5.3 was centered at 5.4, almost a full pH unit higher. These findings confirm that the Zn(II) coordination sphere can be tuned to alter the unfolding pH of molecules related to ZF5.3 and that AV5.3 undergoes a cooperative, pH- and Zn(II)-dependent structural transition with a mid-point that is substantially higher than ZF5.3. AV5.3 was selected for further study here because CCHC’s pH-dependent unfolding transition was virtually identical to that of ZF5.3. Notably, the

[0030] 4 B25-037-2WO unfolding pH of AV5.3 (5.4) corresponds most closely to that of the late endosomal lumen (approximately 5.5), whereas that of ZF5.3 (4.6) is closer the pH of a lysosome.16

[0031]

[0027] AV5.3 traffics efficiently into the cytosol of Saos-2 cells

[0032]

[0028] Next we used confocal microscopy and flow cytometry (FC) to evaluate whether the difference in pH-dependent unfolding of ZF5.3 and AV5.3 was accompanied by a change in overall cell uptake. Variants of ZF5.3 and AV5.3 carrying an N-terminal Nε-azido-L-lysine residue were prepared by solid phase peptide synthesis and fluorescently labeled upon reaction with DBCO-functionalized lissamine rhodamine B to generate AV5.3Rhoand ZF5.3Rho.

[0033] Following purification, AV5.3Rhoand ZF5.3Rhowere added at concentrations between 0.1 and 1 μM to human osteosarcoma (Saos-2) cells and incubated for 30 min. The cells were washed with trypsin to eliminate surface-bound protein and visualized using confocal microscopy.

[0034]

[0029] Confocal microscopy images of Saos-2 cells treated with either AV5.3Rhoor ZF5.3Rhoshow clear evidence of uptake, with substantial punctate fluorescence distributed throughout the cell interior. Quantification of the internalized fluorescence as a function of AV5.3Rhoor ZF5.3Rhoconcentration using flow cytometry indicated that ZF5.3Rhois taken up slightly more efficiently than AV5.3Rho. On average, cells treated with ZF5.3Rhoshowed 2-4-fold higher fluorescence at all treatment concentrations relative to cells treated with AV5.3Rho, with larger differences at higher treatment concentrations. In both cases, whether assessed qualitatively using confocal microscopy or quantitatively using flow cytometry, the uptake of fluorescence was dose-dependent. Overall these studies indicate that conversion of the Cys2His2coordination sphere in ZF5.3 to the Cys4coordination sphere in AV5.3 has a small but measurable effect on overall uptake by Saos-2 cells.

[0035]

[0030] We next made use of fluorescence correlation spectroscopy (FCS) to assess what fraction of the material taken up into the endocytic pathway trafficked successfully into the cytosol. FCS is a unique tool for assessing endosomal escape, as it provides both the precise concentration of a fluorescent molecule in the cytosol as well as its diffusion constant.17We measured the in vitro diffusion constants of ZF5.3Rhoand AV5.3Rhoto determine the cut-offs for an appropriate range of diffusion constants measured in cellula by FCS. FCS analysis of Saos-2 cells treated for 30 min with AV5.3Rhoor ZF5.3Rhorevealed that both molecules reached the cytosol efficiently and in a dose-dependent manner. Treatment of Saos-2 cells with 0.1, 0.5, and 1 μM AV5.3Rholed to average cytosolic concentrations of 59 (± 5), 201 (± 21), and 439 (± 58) nM, respectively, corresponding to delivery efficiencies between 40% and 59%. In comparison, treatment of Saos-2 cells with analogous concentrations of ZF5.3Rholed to average cytosolic concentrations of 69 (± 8), 260 (± 49), and 406 (± 43) nM, corresponding to delivery efficiencies between 41% and 69%. Thus we found no statistically significant difference between the

[0036] 5 B25-037-2WO cytosolic concentrations established in Saos-2 cells treated with equivalent concentrations of AV5.3Rhoor ZF5.3Rho, despite the higher uptake of ZF5.3 detected using flow cytometry. Our results demonstrate that, despite slightly lower uptake, AV5.3 reached the cytosol as well as ZF5.3. This result implies that AV5.3 escapes from the endocytic pathway with higher efficiency than does ZF5.3.

[0037]

[0031] AV5.3 reaches the cytosol in a CORVET- and HOPS-dependent manner

[0032] Previous research has shown that both ZF5.3 and ZF5.3-protein conjugates rely on the HOPS complex for cytosolic access.5,6,8,18–20When cells are depleted of the Rab7-binding HOPS subunits VPS39 and VPS41, the ability of ZF5.3 to reach the cytosol is substantially diminished. The same is true for ZF5.3-protein conjugates that efficiently reach the cytosol.5,6,8In contrast, knockdown of the Rab5-binding CORVET subunits VPS8 and TGF-BRAP1 fails to diminish the ability of ZF5.3 to reach the cytosol. In fact, in some cases, depletion of TGF-BRAP1 improves cytosolic delivery of ZF5.3 and ZF5.3-protein conjugates,5,6,8perhaps because it increases the intracellular concentration of HOPS.21We used analogous siRNA experiments to evaluate the effect of HOPS- and CORVET depletion on the delivery efficiency of AV5.3. If the higher-pH unfolding of AV5.3 leads to escape from an earlier, high-pH endocytic compartment, then escape of AV5.3 should show an higher dependence on CORVET, which operates at an earlier stage of the endocytic pathway.16,22

[0038]

[0033] Saos-2 cells were transfected with siRNAs targeting each of the CORVET-specific subunits VPS8 and TGF-BRAP1 or the HOPS-specific subunits VPS39 and VPS41, and ≥58% knockdown efficiencies established using quantitative PCR. After siRNA transfection, Saos-2 cells were treated with 1 µM AV5.3Rhoor ZF5.3Rhofor 30 min and the cells analyzed using confocal microscopy, flow cytometry, and FCS.

[0039]

[0034] Cells treated with ZF5.3Rhoresponded to HOPS and CORVET depletion in a manner consistent with previous reports.8Depletion of HOPS-specific subunits VPS39 and VPS41 visually decreased the punctate fluorescence evident by confocal microscopy, reduced by 33% the level of internalized ZF5.3Rhofluorescence detected by flow cytometry, and reduced by 60% the concentration of ZF5.3Rhothat reached the cytosol as determined by FCS. Depletion of CORVET-specific subunits also had the expected effects.

[0040]

[0035] Cells treated with AV5.3Rhoresponded differently to HOPS and CORVET depletion than cells treated with ZF5.3. In this case, depletion of either HOPS subunits (VPS39, VPS41) or CORVET subunits (VPS8, TGF-BRAP1) decrease the overall uptake of AV5.3Rhoand its localization to the cytosol. Depletion of HOPS subunits VPS39 and VPS41 decreased the concentration of AV5.3Rhothat reached the cytosol by 86 and 87%, respectively, while depletion of CORVET subunits VPS8 and TGF-BRAP1 led to a decrease of 74 and 92%, respectively. No 6 B25-037-2WO changes in the cytosolic localization of AV5.3Rhowere observed when cells were mock-transfected or transfected with a chemically modified siRNA that fails to engage with the Risc complex (Risc-free). It is notable that the cytosolic localization of AV5.3 is almost completely abolished by either HOPS or CORVET knockdown, suggesting an interplay between these two tethering complexes that is not fully understood. Regardless, these results indicate that both CORVET and HOPS contribute to the cytosolic localization of AV5.3, and that AV5.3 escapes, at least in part, from Rab5+ vesicles that are substrates for CORVET. Moreover, the dependence of AV5.3 delivery on both HOPS and CORVET provides additional support for a mechanistic link between ZF5.3 / AV5.3 unfolding and endosomal escape.9

[0041]

[0036] AV5.3 provides DHFR with an alternate but equally effective path into the cytosol

[0037] Next we sought to determine whether the alternative, HOPS and CORVET-dependent path into the cytosol taken by AV5.3 also supports the delivery of protein cargo. One of the most efficiently delivered cargos when fused to ZF5.3 is dihydrofolate reductase (DHFR), in large part because the DHFR TMis low in the absence of bound ligand.23Samples of AV5.3–DHFR and ZF5.3–DHFR were purified to homogeneity from E. coli and characterized using LC / MS and CD. The effects of pH and the presence of DHFR’s ligand, methotrexate (MTX), on the secondary structure and thermal stability of AV5.3–DHFR and ZF5.3–DHFR were virtually identical. Rhodamine-labeled derivatives of each conjugate (AV5.3–DHFRRhoand ZF5.3– DHFRRho) were prepared using sortase as described previously.5

[0042]

[0038] To evaluate the delivery of protein cargo, Saos-2 cells were treated with between 0.1 and 1 μM AV5.3-DHFRRhoor ZF5.3–DHFRRhofor 1 h and visualized using confocal microscopy, FC, and FCS as described above. Confocal microscopy and FC revealed that AV5.3-DHFRRhoor ZF5.3–DHFRRhowere taken up almost identically by Saos-2 cells and in a dose-dependent manner. The overall uptake of ZF5.3–DHFRRhoby Saos-2 cells is comparable to levels observed previously.5In a similar way, FCS analysis revealed that both AV5.3-DHFRRhoor ZF5.3–DHFRRhoreached the Saos-2 cytosol in a dose-dependent manner and with almost identical efficiency. Finally, knockdown experiments revealed the overall uptake of AV5.3-DHFR as well as its ability to reach the cytosol depends on both the Rab7-binding subunits of HOPS as well as the Rab5-binding components of CORVET. These results confirm that the alternative, HOPS and CORVET-dependent path into the cytosol taken by AV5.3 fully supports the delivery of protein cargo.

[0043]

[0039] Comparing the enzymatic activity of AV5.3-DHFR and ZF5.3-DHFR: in vitro controls

[0044]

[0040] The data presented above support a model in which AV5.3 escapes from the endocytic pathway into the cytosol, at least in part, from earlier, and presumably less acidic endocytic 7 B25-037-2WO compartments than does ZF5.3. This difference should improve the residual activity of delivered proteins or enzymes that struggle to refold and / or regain activity after exposure to low pH. Mammalian DHFR is one such enzyme. Although DHFR refolds after guanidinium hydrochloride-induced denaturation,24it fails to refold after heat treatment or exposure to low pH.5

[0045]

[0041] To evaluate the residual activities of ZF5.3–DHFR and AV5.3–DHFR post-delivery, we first assessed their catalytic activities in vitro in comparison with a human DHFR standard. DHFR catalyzes the reduction of 7,8-dihydrofolate (DHF) to 5,6,7,8-tetrahydrofolate (THF) using a single equivalent of NADPH as a cofactor. Its activity is conveniently measured spectrophotometrically by monitoring the decrease in NADPH absorbance at 340 nm in a reaction mixture supplemented with enzyme and DHF. To evaluate enzyme activities in vitro, solutions of hDHFR, ZF5.3-DHFR, or AV5.3-DHFR at 250 nM were prepared and the enzymatic reaction initiated upon addition of 50 µM DHF.25The resulting decrease in A340was monitored as a function of time and used to calculate specific activity in units of µmole / min / mg protein.

[0046]

[0042] Analysis of the time-dependent decreases in NADPH absorbance revealed that both ZF5.3-DHFR and AV5.3-DHFR are catalytically active. The activity of all three proteins fell between 6.1 and 9.7 moles / min / mg protein. As expected, pre-incubation of hDHFR, AV5.3-DHFR or ZF5.3-DHFR with 2 molar equivalents of methotrexate (MTX) led to a complete loss of enzymatic activity. Thus, when measured in vitro, there was no significant difference between the specific activities of AV5.3-DHFR and ZF5.3-DHFR and no significant difference between either of these two conjugates and hDHFR itself.

[0047]

[0043] Cytosolic DHFR is more catalytically active when delivered by AV5.3

[0048]

[0044] To evaluate the activities of cytosolic ZF5.3-DHFR and AV5.3-DHFR post-delivery, we made use of a commercial CHO cell line that lacks DHFR (CHO / dhFr-). Although DHFR is otherwise essential, CHO / dhFr- cells remain viable and grow upon addition of hypoxanthine and thymidine to compensate for the absence of endogenous DHFR. We prepared cytosolic extracts of CHO / dhFr- cells both before and after treatment with ZF5.3-DHFR or AV5.3-DHFR, and assessed the residual DHFR activity as described above. Cytosolic extracts of CHO / dhFr- that had not been treated with ZF5.3-DHFR or AV5.3-DHFR showed no detectable DHFR activity, as expected. Supplementing these non-treated extracts with 250 nM hDHFR restored DHFR activity to a value of 2.34 moles / min / mg protein; this activity was abolished in the presence of 500 nM MTX.

[0049]

[0045] Next we treated live cultures of CHO / dhFr- cells with 1 µM of either AV5.3-DHFR or ZF5.3-DHFR for 1 h, prepared cytosolic extracts, and tested the extracts for DHFR activity.

[0050] 8 B25-037-2WO Cytosolic extracts of CHO / dhFr- cells treated with 1 µM AV5.3-DHFR were characterized by a DHFR activity of 2.0 moles / min / mg, 15% lower than the activity observed when untreated extracts were supplemented with 250 nM hDHFR. This value is only slightly lower than the concentration of AV5.3-DHFR that reaches the Saos-2 cytosol after a 1 µM treatment (329.1 ± 36 nM). This result suggests that AV5.3-DHFR retains significant activity even after exposure to the endocytic pathway. In contrast, cytosolic extracts of CHO / dhFr- cells treated with 1 µM ZF5.3-DHFR were characterized by a DHFR activity of 0.5 moles / min / mg, a value that is roughly 80% lower than the activity observed when untreated extracts were supplemented with 250 nM hDHFR, and equally lower than the concentration of ZF5.3-DHFR that reaches the Saos-2 cytosol after a 1 µM treatment (291.2 ± 22 nM). The stark difference between the residual activities of AV5.3-DHFR or ZF5.3-DHFR in cytosolic extracts post-delivery provides direct evidence that AV5.3 not only delivers protein cargos efficiently but also provides confidence that even highly pH-sensitive cargo proteins and enzymes will retain activity upon reaching the cytosol.

[0051]

[0046] AV5.3-DHFR rescues the DHFR deficiency of CHO / dhFR- cells

[0052]

[0047] Finally we asked whether AV5.3-DHFR would rescue, in full or in part, the DHFR deficiency of CHO / dhFR- cells. CHO / dhFR- cells fail to grow without addition of hypoxanthine and thymidine to enable DNA biosynthesis, and their viability, as measured by ATP activity, diminishes slowly over the course of 72 h. Supplementation every 12 h with 100 µM hypoxanthine and 16 µM thymidine fully maintains cell viability as measured by the concentration of ATP (CellTiter-Glo® 2.0 Cell Viability Assay). When the concentration of hypoxanthine and thymidine was reduced by half, cell viability decreases by roughly 50% over 48 h. Treatment of CHO / dhFR- cells with 1 µM ZF5.3-DHFR had no significant effect on cell viability in the presence or absence of hypoxanthine and thymidine supplement. In contrast, CHO / dhFR- cells treated with 1 µM AV5.3-DHFR remained viable over the course of 72 h in the presence or absence of hypoxanthine and thymidine supplement. In the absence of any supplement, viability of AV5.3-DHFR-treated cells after 72 h was roughly 35% lower than the viability of untreated CHO / dhFR- in the presence of complete hypoxanthine and thymidine supplement. In the presence of half strength supplements, cells treated with 1 µM AV5.3-DHFR were only 16% less viable than untreated CHO / dhFR- in the presence of complete hypoxanthine and thymidine supplements. In the presence of full strength hypoxanthine and thymidine supplement, CHO / dhFR- cells treated with 1 µM AV5.3-DHFR showed almost 25% greater proliferation than cells lacking AV5.3-DHFR treatment. We conclude from these data that the residual enzyme activity of AV5.3-DHFR upon delivery to the cytosol is sufficient to rescue a genetic DHFR deletion in CHO cells.

[0053] 9 B25-037-2WO

[0048] Some Conclusions

[0054]

[0049] This project was initiated to overcome one of the major challenges facing any delivery strategy that relies on the endocytic pathway: exposure of cargo to low pH. Using insights from mechanistic studies on the pathway of endosomal escape and classic knowledge regarding the bioinorganic chemistry of zinc(II) coordination, we re-designed the sequence of a known mini-protein to successfully alter the timing of endosomal escape. When this new mini-protein, AV5.3, is conjugated to an acid-labile enzyme cargo, dihydrofolate reductase (DHFR), delivery efficiency is unaffected but the residual activity of DHFR in the cytosol is substantially improved. Mutations in mammalian DHFR lead to dihydrofolate reductase deficiency,26a rare autosomal recessive inborn error of metabolism.26DHFR deficiency could be treated using replacement enzyme therapy, but only if the delivered enzyme can reach internal cellular locales and retain activity.27Here we show that endosomal escape can be fine-tuned to improve the residual activity of a mammalian enzyme that traverses through the endocytic pathway to reach the cytosol. This work shows that programmed trafficking through the endosomal pathway is a viable strategy for efficient cytosolic delivery of therapeutic proteins. It also provides de facto support that endosomal escape of mini-proteins like ZF5.3 and AV5.3 demand protein unfolding in acidic endosomal compartments.9

[0055]

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[0095]

[0051] Table 1: Peptide, Protein, and DNA / RNA Sequences

[0096] Solid-Phase Peptide Sequences

[0097]

[0098]

[0099] 13 B25-037-2WO RISC-free siGENOME RISC-Free Control siRNA: D-001220-01 (Dharmacon)

[0100]

[0101] 14 B25-037-2WO

Claims

CLAIMS1. An engineered endosomal escape polypeptide domain comprising a ZF5.3 sequence:YSCNVCGKAFVLSRHLNRHLRVHRRAT (SEQ ID NO:01) having Cys substitutions of one or both His residues of the Cys2His2Zn(II) coordination site, to form a Cys3His (CCHC) or Cys4(AV5.3) a coordination site, respectively.

2. An engineered endosomal escape polypeptide domain comprising the sequence:YSCNVCGKAFVLSRHLNRHLRVCRRAT (SEQ ID NO:03) (CCHC).

3. An engineered endosomal escape polypeptide domain comprising the sequence:YSCNVCGKAFVLSRHLNRCLRVCRRAT (SEQ ID NO:02) (AV5.3).

4. An endosomal escape polypeptide domain of claim 2, covalently or noncovalently bound to a cargo (e.g. a bioactive molecule, label, nucleic acid and polypeptide), wherein the endosomal escape polypeptide domain facilitates endosomal escape to improve cytosolic delivery of the cargo.

5. An endosomal escape polypeptide domain of claim 3, covalently or noncovalently bound to a cargo (e.g. a bioactive molecule, label, nucleic acid and polypeptide), wherein the endosomal escape polypeptide domain facilitates endosomal escape to improve cytosolic delivery of the cargo.

6. An endosomal escape polypeptide domain of claim 2, covalently bound to a cargo (e.g. a bioactive molecule, label, nucleic acid and polypeptide), wherein the endosomal escape polypeptide domain facilitates endosomal escape to improve cytosolic delivery of the cargo.

7. An endosomal escape polypeptide domain of claim 3, covalently bound to a cargo (e.g. a bioactive molecule, label, nucleic acid and polypeptide), wherein the endosomal escape polypeptide domain facilitates endosomal escape to improve cytosolic delivery of the cargo.

8. An endosomal escape polypeptide domain of claim 2, covalently bound to a cargo polypeptide, wherein the endosomal escape polypeptide domain facilitates endosomal escape to improve cytosolic delivery of the cargo.15 B25-037-2WO9. An endosomal escape polypeptide domain of claim 3, covalently bound to a cargo polypeptide, wherein the endosomal escape polypeptide domain facilitates endosomal escape to improve cytosolic delivery of the cargo.

10. A lipid nanoparticle delivery vehicle for delivering a cargo (e.g. a bioactive molecule, label, protein, nucleic acid, or complexes thereof) to a cytosol, comprising a lipid nanoparticle comprising an endosomal escape polypeptide domain of any of claims 4-9.

11. A pharmaceutical composition comprising an endosomal escape polypeptide domain of any of claims 1-9 and a pharmaceutically acceptable excipient, in effective, unit dosage.

12. A method of delivering a cargo to a cytosol, comprising delivering to a cell a composition comprising an endosomal escape polypeptide domain of any of claims 4-9, under conditions wherein the endosomal escape polypeptide domain facilitates endosomal escape of the cargo to the cytosol.

13. A method of delivering a cargo to a cytosol, comprising delivering to a cell a composition comprising an endosomal escape polypeptide domain of any of claims 4-9, under conditions wherein the endosomal escape polypeptide domain facilitates endosomal escape of the cargo to the cytosol, and further comprising a subsequent step of detecting the delivery of the cargo to the cytosol.

14. A method of treating a disease or disorder or condition in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a composition comprising an endosomal escape polypeptide domain of any of claims 1-9.

15. A method of treating a disease or disorder or condition in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a composition comprising an endosomal escape polypeptide domain of any of claims 1-9, and further comprising an antecedent step of detecting the disease, disorder or condition, and further comprising a subsequent step of detecting a resultant effect or improvement in the disease, disorder or condition.16 B25-037-2WO

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